Physicochemical methods (direct introduction of DNA), such as a polyethylene glycol method, an electroporation method, and a particle gun method, and biological methods (indirect introduction of DNA) utilizing functions of Agrobacterium are known as methods for transformation of monocotyledons such as barley, wheat, corn, and rice, which are major cereal crops. The direct introduction, however, frequently causes introduction of a fragmented objective gene or introduction of multiple copies of an objective gene. As a result, a transformant that does not express the objective gene or shows abnormal weak expression (gene silencing) appears at a high frequency. A method using a protoplast needs a prolonged culture period, which tends to cause seed sterility or malformation in the resulting transformant due to variation during the culture.
In contrast, in the gene introduction mediated by Agrobacterium, for example, the regulation of expression of gene groups in a Ti plasmid virulence region (vir region) maintains the small number of copies of an objective gene and prevents a gene from being introduced as fragmented segments. The gene introduction mediated by Agrobacterium therefore has notable advantages of providing a large number of transformants highly expressing the objective gene and allowing the difference in expression levels of individual transformants to be small, compared to the direct gene introduction.
Gene introduction mediated by Agrobacterium is generally used for transformation of dicotyledons. Although it has been believed for a long time that hosts of Agrobacterium in nature are limited only to dicotyledons and Agrobacterium has no ability to infect monocotyledons (Potrykus 2000: Non-Patent Literature 1), a method of transformation of a monocotyledon by Agrobacterium at a high efficiency has been first reported in major cereal crops, rice, as a result of detailed studies such as investigation of tissue materials, improvements in medium compositions, and selection of Agrobacterium strains (Hiei et al., 1994: Non-Patent Literature 2). Following on the success in rice, examples of successful transformation mediated by Agrobacterium in corn (Ishida et al., 1996: Non-Patent Literature 3), wheat (Cheng et al., 1997: Non-Patent Literature 4), barley (Tingay et al., 1997: Non-Patent Literature 5), and sorghum (Zhao et al., 2000: Non-Patent Literature 6) have been reported. As materials of Agrobacterium-mediated transformation in monocotyledonous crops, immature embryos and immature embryos cultured for a short period of time are most appropriate, and in crops such as corn, wheat, and barley, immature embryos are main targets of Agrobacterium infection (Cheng et al., 2004: Non-Patent Literature 7).
The first successful example of transformation by Agrobacterium in barley is also a method using immature embryos as the material (Tingay et al., 1997: Non-Patent Literature 5). As concretely described below, the methods for transformation of barley that have, been recently reported (Jacobsen et al., 2006: Non-Patent Literature 8; Bartlett et al., 2008: Non-Patent Literature 9; Hensel et al., 2008: Non-Patent Literature 10; and Harwood et al., 2008: Non-Patent Literature 11) are also basically the same as the method by Tingay et al. (1997: Non-Patent Literature 5). Though the method by Tingay et al. (1997: Non-Patent Literature 5) includes wounding an immature embryo with a particle gun prior to inoculation with Agrobacterium, the wounding treatment is rarely performed since Trifonova et al. (2001: Non-Patent Literature 12) has shown that the wounding treatment of a barley immature embryo with a particle gun prior to inoculation with Agrobacterium does not increase the transformation efficiency.